Food Safety Monitoring of Salmonella spp. in Northern Italy 2019–2021

Salmonella is the second most frequent bacterial pathogen involved in human gastrointestinal outbreaks in the European Union; it can enter the food-production chain from animal or environmental sources or from asymptomatic food operators. European food legislation has established microbiological criteria to ensure consumer protection. Salmonella is listed under both process hygiene criteria and food safety criteria. Each EU member state designates an agency to organize or perform controls and other official activities. This paper describes the official control plans performed by competent authorities in Northern Italy in the three-year period 2019–2021. A total of 4413 food samples were delivered to the IZS Food Safety laboratories for Salmonella detection, of which 36 (0.8%) tested positive. Salmonella was most frequently detected in poultry meat samples (25/36 positive samples) followed by other meat products and pork products. The official controls for the protection of consumer health apply the EU’s farm-to-fork approach: the samples were collected during production (food production plants), from products on the market, and from collective catering (restaurants, cafeterias, canteens). This manuscript will provide information about the presence of Salmonella in foodstuffs that can help competent authorities to set control plans based on risk assessments.


Introduction
Salmonella is a Gram-negative, facultative anaerobe bacterium that inhabits the intestines of humans and animals [1]. Wild birds can be reservoirs as can domestic animals [2]. Salmonella can also be found in water, the environment, and contaminated food products [1].
Salmonella is the second most frequent bacterial pathogen involved in human gastrointestinal outbreaks in the European Union, particularly S. Enteritidis and S. Typhimurium [4].
Food safety agencies can make use of official laboratories for accredited analysis of food samples. In Italy, the Istituti Zooprofilattici Sperimentali (IZS) form the national network of laboratories that provide scientific support and chemical and microbiological analyses for food safety agencies and other control bodies.
This article describes the work of the food safety laboratory of the IZS, Turin, and reports the results of Salmonella detection analysis of food products collected during official monitoring of the food chain of animal and vegetable origin for the three-year period 2019-2021. The data reported here refer to the epidemiological situation in northwestern Italy, which comprises the three regions of Piedmont, Liguria, and Valle d'Aosta. The official laboratory for food safety analytical control for this area is the Istituto Zooprofilattico Sperimentale del Piemonte Liguria and Valle d'Aosta, a public health agency under the Ministry of Health and accredited according to international standards for organizational procedures.

Sampling
Food samples were collected in the context of official food safety monitoring by local health services (Piedmont, Liguria, Valle d'Aosta). All samples were collected using sterile instruments according to instructions for obtaining samples and for transport to the laboratory, as described in international standard protocols [16]. The food samples were delivered to the IZS laboratories at the temperature reported on the label by food business operators for correct conservation of the particular foodstuff and analyzed within 24 h after arrival at the lab.

Rapid Screening Methods
Detection of Salmonella spp. was performed by rapid screening methods validated according to ISO 16140:2021 [17] and accredited (Table 1).

Microbiological Methods
Isolation and identification of Salmonella spp. were performed according to ISO 6579 [18] using the media described in international standard protocols ( Table 2). Five suspected colonies from each Petri dish were streaked on Columbia blood agar (CBA) (Becton & Dickinson, Franklin Lakes, NJ, USA) and incubated at 35 • C for 24 ± 2 h. One isolated colony from each CBA plate was analyzed with a MALDI Biotyper ® Sirius System mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) using the extended direct transfer (eDT) procedure. One loop of biomass was transferred to a MBT96 polished steel BC target plate (Bruker Daltonik GmbH) spot. The air-dried sample spot was overlaid with 1 µL of a formic acid water solution (70% v/v) and, after air-drying, with 1 µL of matrix HCCA (α-cyano-4-hydroxycinnamic acid) solution (Bruker Daltonik GmbH). Target plates of the samples were analyzed, and spectra were acquired in positive ion mode in the m/z range 2000-20,000. As an alternative, one isolated colony from each CBA plate was streaked on triple sugar iron agar (TSI) (Biolife Italiana S.r.l., Milan, Italy). Positive TSI colonies were confirmed by biochemical analysis using API 20E galleries (bioMérieux, Marcy l'Étoile, France).

Serotyping
The strains confirmed as being S. enterica were subcultured on Columbia blood agar (Becton & Dickinson) at 37 • C for 24 h and then serotyped according to the Kaufmann-White-Le Minor scheme [19] using O and H antisera (Statens Serum Institut, Artillerivej, Denmark).

Statistical Analysis
After cleaning and preparing the data, descriptive statistical analysis was conducted to obtain information about the distribution of Salmonella detection analyses in food matrices, criteria, and sampling sites monitored during the three-year period and to investigate for a possible seasonal effect. Prevalence and confidence intervals were calculated using Stata Special Edition, release 17.1 (StataCorp, College Station, TX, USA).

Samples Delivered to the Laboratories
A total of 4413 food samples were delivered to the IZS food safety laboratories for Salmonella detection during the three-year period 2019-2021. Three main sampling plans were in operation: (1) official monitoring of national food products (n = 4222); (2) official monitoring of imported/exported food products (n = 126); (3) official monitoring of imported/exported food products after previous reports of non-compliance (n = 65).
In 2019, 1548 samples were collected and analyzed; in 2020, 1404 samples; and in 2021, 1461 samples. Table 3 (Table 3). The entire food production and serving chain undergoes official monitoring by a competent agency. Sampling is conducted on foodstuffs falling under the responsibility of food business operators (in production plants) or on foodstuffs for sale on the market or at collective catering outlets (Table 4). Food matrices were collected according to the regional plans for food safety based on risk assessment studies of food categories (Table 5). * Non-poultry meat and poultry meat are fresh, refrigerated, unprocessed meat products; a meat product is a multi-ingredient food product in which meat is the main ingredient.

Detection of Salmonella spp.
Out of the total of 4413 food samples collected and analyzed, 36 (0.8%) tested positive for Salmonella spp. Out of the total of 3328 samples collected on the market, 33 (0.99%) tested positive and 3 out of the total of 1030 (0.29%) collected at a production plant tested positive.
Of the food samples collected during monitoring of national foodstuff products (n = 4222), 30 tested positive (30/4222; 0.71%). The rates of positive samples collected during official monitoring of imported/exported food products and imported/exported food products collected for testing after previous results were non-compliant were 1/126 (0.79%) and 5/65 (7.69%), respectively.
Salmonella spp. was most frequently detected in poultry meat samples (25/36 positive samples) followed by other meat products and pork products. The remaining Salmonella strains were found in fishery samples, a raw-milk cheese sample, and in bulk cereals from a third country ( Table 6). The percentage of positive samples by year ranged between 0.45% (2019) and 1.64% (2020) ( Table 7). Non-poultry meat samples testing positive were pork meat; the positive dairy sample was a raw-milk cheese; fishery products testing positive were Gallus provincialis (n = 1) and Mytilus galloprovincialis (n = 1); and a sample of soy seeds in the cereals and seeds category was also found to be positive. Table 8 presents the Salmonella serovars according to the Kauffman-White scheme. Salmonella Infantis was the most frequently isolated and identified serovar (n = 19), followed by S. Derby (n = 3), S. Enteritidis (n = 3), and the monophasic variant S. Typhimurium 4,5,12;i;-; (n = 3). S. Infantis and S. Enteritidis were most often isolated from poultry meat, while S. Derby and S. Typhimurium were most often isolated from pork products. Other serovars were identified in one sample; one strain isolated from a fish product (Gallus provincialis) was classified as group O:11; H: e,h,x and could not be assigned to a specific serovar.

Seasonality of Salmonella Detection
The multivariable analysis investigated for a possible seasonal effect. The only time covariate that significantly associated with the detection of Salmonella spp. was the year 2020 as the time of data collection and analysis. Table 9 presents the prevalence ratio (PR) of exposed (year 2020) versus non-exposed (year 2019). The covariate was statistically significant: 1 not included in the 95% confidence interval.

Discussion
According to the online EFSA dashboard [20], between 2017 and 2021, sampling units from five food categories from 37 countries (29 EU member states) were tested for Salmonella The high incidence of S. Infantis detected in poultry meat is shared by the 2019 report published by the National Reference Center for Salmonellosis. In 2019, S. Infantis was the most often detected serotype in Gallus gallus farms and 44% of S. Infantis strains were detected in broilers [21]. S. Infantis was massively reported for broiler matrices in the EU in 2019, from animals (36.3% of all serotyped isolates), and from other food matrices (49.1%). More than 50% of the S. Infantis isolates from broilers in 2019 were reported for Italy. In addition, in 2020, S. Infantis and S. Derby isolates were most often reported for Italy, which accounted for 43% and 38.3%, respectively, of the isolates positive for these serovars [5]. Broiler meat is a common source of Salmonella, and the contamination of broiler farms has been increasingly associated with persistent serovars, such as S. Infantis [7].
Considering human cases of nontyphoid salmonellosis in northwestern Italy, the most often detected serovars in the period 2017-2021 were the monophasic variant S. Typhimurium 4,5,12;i;-(45.5%), S. Typhimurium (13.8%), and S. Enteritidis (9.4%). The average proportion of human cases caused by S. Infantis during the same period was 1.7%, with a rise from 0.5% in 2017 to 2.5% in 2021 [22]. The increase in the proportion of human cases attributable to S. Infantis is consistent with the data reported for food samples and for broiler farms from the National Reference Center for Salmonellosis.
A certain seasonality for Salmonella detection has been suggested. Salmonellosis may be caused by the convergence of different factors, including human behavior, prevalence in animal reservoirs, consumption patterns, and bacterial environmental survival. Variation in seasonal prevalence seems to be greater during warmer months and lower during colder months [23]. The multivariable Poisson regression model we used to investigate for a time effect revealed that the covariates representing the season in which the sample was collected and analyzed were not statistically significant. The observation period was quite short and other variables related to the COVID-19 pandemic might have made it difficult to identify an expected seasonal trend. The only significant time-related covariate was the difference in rates recorded for 2020 and 2019: the PR reflects the high proportion of Salmonella spp. positive samples in 2020 versus 2019 (3.62 times higher). Nontyphoidal salmonellosis, such as S. Enteritidis, S. Newport, and S. Typhimurium serotypes, are frequently associated with foodborne disease outbreaks from contaminated eggs, meat, milk products, and poultry [24].
The safety monitoring plans for foods of animal and vegetable origin are carried out at the local level and based on the number of food-producing plants located in the area and the size of the resident population. The IZS covers all of northwestern Italy, which includes Piedmont (about 4.3 million inhabitants), Liguria (about 1.5 million inhabitants), and Valle d'Aosta (about 125,000 inhabitants). Considering the differences in regional population density, the number of samples collected per region appears to be consistent. Furthermore, the number of analyses carried out in the three regions was constant during the three-year period for the total number of samples collected and for the type of matrix delivered to the laboratory.
The IZS laboratories use rapid screening methods to detect food-related pathogens, with negative results obtained in less than 24 h from the start of analysis (less than 48 h after collection): this is essential in the monitoring of fresh and perishable food. If the lab test result is non-compliant with established legal limits, food health officers undertake procedures to withdraw and recall food products considered unsafe according to Regulation (EU) 178/2002, art. 14 [25].
Monitoring of food imports from third countries entails a series of random samplings. The three-region positivity rates for Salmonella spp. were similar to the national rates in official controls; however, following reports of non-compliance at the time of importation into Europe through an Italian border point, subsequent analytical checks are performed to protect consumer health in EU member states and to prevent entry of potentially harmful food products into the EU.
In the context of official monitoring, controls following previous non-compliance reports play a vital role in protecting consumer health. Detection of unhygienic practices at food plants or in relation to certain food products is followed up with subsequent checks, as confirmed by these data. The food safety agencies perform serial controls to investigate the source of contamination and to determine whether corrective actions implemented by food business operators are truly effective.
Our data show a higher percentage of Salmonella positivity in food samples recorded for 2020. Food safety monitoring and sampling continued to protect consumer health during the COVID-19 pandemic. The relatively higher percentage of positives is not statistically significant, and there are currently no published data to compare similar trends for other EU countries or other food pathogens. The economic losses and difficulties in operations management experienced by food business operators because of the pandemic restrictions may have affected food safety standards in supply chains and production plants. This issue is awaiting consideration by other working groups and laboratories involved in food safety monitoring.